Project Summary The long-term goal of this work is to elucidate, in molecular detail, the in vivo signaling mechanisms of the transmembrane receptors that mediate chemotactic behavior in E. coli, with predominant focus on Tsr, the serine chemoreceptor. Tsr and other chemoreceptors form stable ternary complexes with two cytoplasmic proteins: CheA, a histidine autokinase, and CheW, which couples CheA to chemoreceptor control. Most of the prodigious signal amplification that occurs in the chemotaxis pathway takes place in these complexes, which are organized into highly cooperative arrays. The overall objectives of the next project period are to extend and test a dynamics-based model of signal transmission through a receptor dimer and to characterize the signaling and clustering properties of receptor molecules that have different dynamic behaviors and clustering abilities. Our working model proposes that the receptor's kinase-activating output emanates from molecules with intermediate structural stabilities and dynamic behaviors, rather than a discrete conformational state. In contrast, receptor molecules at either extreme of the dynamic range, i.e., ones whose structures are too "molten" or too "frozen", are proposed to adopt kinase-deactivating states. We expect that the proposed studies will clarify the nature of the kinase-on and kinase-off signaling states of chemoreceptors, which in turn will permit development of more incisive mechanistic models for kinase control in receptor signaling complexes. Moreover, our experiments will serve to identify the structural and functional features of receptor molecules that are most critical for assembly of receptor signaling complexes and clustered arrays. Finally, the proposed studies will determine which receptor properties are important to their highly cooperative signaling behavior and whether such signaling is accompanied by changes in the spatial organization of receptor arrays. The proposed studies have three specific aims: 1. Elucidate the mechanisms of conformational coupling between the HAMP domain and its adjoining input and output elements. 2. Assess the dynamic properties of chemoreceptor molecules and test predictions of dynamics-based signaling models.. 3. Characterize the signaling and clustering properties of mutant receptors with more quantitative and higher resolution methods. PUBLIC HEALTH RELEVANCE: Project Narrative Chemotactic behaviors influence the environmental distributions of motile microorganisms, the composition of microbial communities such as biofilms, and host invasion during the establishment of beneficial symbioses and harmful infections. Better understanding of the molecular mechanisms of stimulus detection and sensory signaling by bacterial chemoreceptors should lead to new strategies for augmenting the beneficial behaviors of bacteria and to new therapies for harmful bacterial infections.